Sustained neuronal activity demands a rapid resupply of synaptic vesicles to maintain reliable synaptic transmission. Such vesicle replenishment is accelerated by submicromolar presynaptic Ca2+ signals by an as-yet unidentified high-affinity Ca2+ sensor1,2. Here we identify synaptotagmin-3 (SYT3)3,4 as that presynaptic high-affinity Ca2+ sensor, which drives vesicle replenishment and short-term synaptic plasticity. Synapses in Syt3 knockout mice exhibited enhanced short-term depression, and recovery from depression was slower and insensitive to presynaptic residual Ca2+. During sustained neuronal firing, SYT3 accelerated vesicle replenishment and increased the size of the readily releasable pool. SYT3 also mediated short-term facilitation under conditions of low release probability and promoted synaptic enhancement together with another high-affinity synaptotagmin, SYT7 (ref. 5). Biophysical modelling predicted that SYT3 mediates both replenishment and facilitation by promoting the transition of loosely docked vesicles to tightly docked, primed states. Our results reveal a crucial role for presynaptic SYT3 in the maintenance of reliable high-frequency synaptic transmission. Moreover, multiple forms of short-term plasticity may converge on a mechanism of reversible, Ca2+-dependent vesicle docking.
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We thank C. Dean for providing Syt3 KO mice and virus plasmids; the OHSU Molecular Virology Core for packaging AAV vectors; K. Wright, K. Monk, S. Kaech Petrie and the staff at the Advanced Light Microscopy Core for imaging assistance; G. Westbrook, H. von Gersdorff, L. Trussell, M. Freeman and P. Brehm and members of the Jackman Lab for comments on the manuscript; J. Vazquez for illustrations; P. Kaeser and myriad colleagues from Vollum for reagents, technical assistance and discussions. This work was supported by the Whitehall Foundation (S.L.J), the Medical Research Foundation (S.L.J) and the NIH Imaging Core Facility (P30NS061800).
The authors declare no competing interests.
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Extended data figures and tables
a-b) Representative fluorescent images showing immunolabeling for SYT3 and VGLUT1 in the brainstem (a) and cerebellum (b) of one WT and one Syt3 KO animal. Representative images were acquired using tissue from a single animal of each genotype, but similar results were obtained using tissue from >5 animals of each genotype while optimizing immunohistochemistry.
a) Representative fluorescent images of calyces of Held from a WT mouse, stained using primary antibodies against SYT3, VGLUT1, and Bassoon, followed by secondaries for all primaries. b-d) Representative images of calyces, where secondaries were applied after tissue was treated with only one primary antibody against either SYT3 (b), VGLUT1 (c), or Bassoon (d). Aside from the omission of primary antibodies, all slices were imaged using the same microscope settings and processed identically. Representative images were acquired using tissue from a single WT mouse, but similar results were obtained from 2 or more animals while optimizing immunohistochemistry.
a) Representative fluorescence images of a calyx of Held from a WT mouse, immunolabeld using primary antibodies against Bassoon, followed by multiple secondaries with different fluorophores. To ensure proper SIM microscope channel alignment, XY displacement between channels was determined, and this shift was used to perform post hoc alignment of images for localization in Fig. 1. b) Inset from a showing overlap of channels following post hoc channel alignment. Active zone fluorescence profiles were assessed using a 100 nm-wide rectangle drawn through the center of Bassoon-labeled puncta, extending from the presynaptic terminal to the postsynaptic cell. c) Averaged fluorescence profiles of Bassoon puncta imaged in 3 channels, showing the full-width at half maximum (FWHM) of gaussian fits to each channel, and the relative offset of fits to fluorescent peaks. (N = 100 puncta from 4 calyces). The increase in FWHM with fluorophore wavelength is not expected to affect the localization analyses presented in Fig. 1. d) Representative calyx of Held from a WT mouse, immunolabelled for SYT3 and PSD-95 (for analysis in Fig. 1c). Data are mean ± s.e.m. Error bands are obscured by the mean. SIM images used for fluorescence profiles were acquired using tissue from one WT animal, but similar results were obtained from 2 or more animals of each genotype while optimizing immunohistochemistry.
a) Representative spontaneous excitatory postsynaptic currents (sEPSCs) recorded from MNTB neurons in WT (black) and Syt3 KO animals (magenta). b) Averaged sEPSC waveforms. c) Rise (P = 0.87) and decay time constants (P = 0.11) of sEPSCs. d) Average (P = 0.41) and cumulative distribution (P = 0.51) of sEPSC amplitudes. e) Average (P = 0.78) and cumulative distribution (P = 0.86) of sEPSC frequency. f) Representative EPSCs elicited by afferent fibre stimulation of the calyx of Held. g) Average rise (P = 0.79) and decay (P = 0.91) time constants, and amplitudes (P = 0.82) of EPSCs. h) Schematic showing presynaptic Ca2+-current (ICa) recordings at the calyx of Held. i) ICa elicited by 1 ms depolarizations from −80 mV to 0 mV at 100 Hz. j) Average amplitudes of initial ICa (P = 0.5). k) Normalized ICa evoked by 20 depolarization steps at 100 Hz. Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Significances were tested using Kruskal-Wallis tests (c,g (rise time and EPSC amplitude)) and two-tailed Student’s t-tests (d, g (τ), j and k). Cumulative distributions in d and e were tested using Kolmogorov-Smirnov tests.
a) Averaged cumulative amplitudes of EPSCs elicited by 200 Hz stimulation at the calyx of Held in WT (black) and Syt3 KO (magenta) animals. Back-extrapolation from EPSC81-10028 corrected for vesicle replenishment early in the train18 was used to estimate the RRPTrain (P = 0.02). b) Amplitudes of the first 40 EPSCs at 200 Hz stimulation plotted against cumulative release. Linear forward-extrapolation of the first 4 EPSCs was used to estimate the RRPEQ19 (P = 0.006). c) Normalized amplitudes of the first 20 EPSCs at 200 Hz stimulation. τDecay was calculated with a single exponential fit. RRPDecay was estimated from τDecay20 (P = 0.03). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as *: P < 0.05, **: P < 0.01. Significances were evaluated using Kruskal-Wallis tests.
a) Illustrated model of vesicle docking and fusion based on previous studies55,56 (see Methods). An infinite pool of reserve vesicles reversibly transitions to a fusion-incompetent loosely docked state. Loosely docked vesicles reversibly transition to a fusion-competent tightly docked state, and release with a fixed probability (p). For the conceptual basis of this model, see21. b) Best fit of the model (magenta) to Syt3 KO data (light magenta) for EPSC trains (left), steady-state amplitudes for frequencies from 1-200 Hz (center), and recovery from depression after 100 EPSCs at 200 Hz (right). c) Illustration of a model where SYT3 reduces vesicle depletion by permanently lowering p, as has been suggested for other synaptotagmin isoforms60. d) Best fit of the model shown in c (black) to data (gray) for WT calyces of Held. e) A model where SYT3 increases p immediately after each action potential, after which p decreases exponentially back to its baseline value. f) Best fit of the model shown in e to WT data. g) A model where SYT3 transiently increases k1. After each action potential k1 increases by a fixed amount, after which k1 decreases exponentially back to its baseline value. h) Best fit of the model shown in g to WT data. i) A model where SYT3 transiently increases k2. After each action potential k2 increases by a fixed amount, after which k2 decreases exponentially back to its baseline value. j) Best fit of the model shown in i to WT data. Note that this model produced the lowest χ2 values among all models tested, supporting a scenario where SYT3 accelerates the transition of vesicles to a tightly docked state. Data were reproduced from Fig. 2. Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1.
a) Ca2+-dependent model of vesicle docking and fusion. The transition from loose to tight docking was accelerated by SYT3 binding to residual calcium ([Ca2+]res). Tightly docked vesicles fused via 5 cooperative binding steps58 that were driven by local calcium ([Ca2+]local) (see Methods). b) Best fit of the model (dark lines) to WT (grey) and KO (light magenta) data. c) Simulated Ca2+-dependent membrane binding by different SYT isoforms driven by an action potential evoked increase in [Ca2+]res. The fraction of active SYT isoforms was governed by their reported Ca2+ affinity and binding kinetics 4,25,61. χ2 values show best fits to WT data for models where each SYT isoform increased docking rates. d) Modified version of the model in a where an additional [Ca2+]local-dependent mechanism was introduced to increase the docking rate (klocal). Because Munc13-1 has been shown to accelerate vesicle priming at multiple synaspes30,31, including the calyx of Held29,59, we modeled this additional mechanism using the Ca2+-binding properties reported for Munc13-162. e) Best fit of the model in e to WT and KO data. The improved model fit to Syt3 KO data supports a role for additional mechanisms such as Munc13-1, in promoting the transition of vesicles to tight docking. Data were reproduced from Fig. 2. Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1.
Extended Data Fig. 8 SYT3 does not affect quantal parameters or Ca2+-dependence of release from cerebellar climbing fibres.
a) Superimposed recordings of 10 climbing fibre EPSCs during 0.1 Hz stimulation in varying [Ca2+]e. b) Average EPSC amplitudes at varying [Ca2+]e concentrations. c) Average variance of EPSCs plotted against mean EPSC amplitude across varying [Ca2+]e concentrations. Parabola fits were used to estimate quantal parameters54 (q: P = 0.76; N: P = 0.38; p: P = 0.78). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances were evaluated using two-tailed Student’s t-tests (b,c (EPSC variance, q, and p)) and Kruskal-Wallis tests (c(N)).
Extended Data Fig. 9 Recovery from depression is slowed by loss of Syt3 at cerebellar mossy fibre synapses.
a) Schematic of cerebellar mossy fibre recordings. EPSCs were recorded from voltage-clamped granule cells (GC) while mossy fibres (MF) were activated by electrical stimulation in the white matter 50-100 μm from the cell. b) Representative EPSCs elicited by 100 stimuli at 200 Hz, followed by stimuli at varying intervals to probe recovery from depression (ΔtRecovery). c-d) Time course of recovery of EPSCs in the first 0.5 s after trains (c, linear scale) and 10 s (d, logarithmic scale). e) Weighted time constants of biexponential recovery (τw) (P = 0.01). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as *: P < 0.05. Significances were evaluated using two-tailed Student’s t-tests.
Extended Data Fig. 10 Recovery from depression is not affected in Syt7 KOs at calyx of Held or climbing fibre synapses.
a) Representative EPSCs elicited by 100 stimuli at 200 Hz, followed by stimuli at varying intervals to probe recovery from depression (ΔtRecovery) at the calyx of Held in WT (black) and Syt7 KO (red) synapses. b) First second of recovery of EPSCs after 200 Hz stimulation, fit with biexponential curves. c) Full time-course of recovery of EPSCs after 200 Hz stimulation. d) Weighted time constant of biexponential recovery (τw) for both genotypes (P = 0.40). e) Superimposed recordings of EPSCs evoked by pairs of stimuli of climbing fibre synapses with varying ΔtRecovery. f-g) Time-course of recovery from depression in linear (f) and logarithmic scale (g). h) Weighted time constant of biexponential recovery (τw) for both genotypes (P = 0.14). WT data were reproduced from Figs. 2 & 4. Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Significances were evaluated using Kruskal-Wallis tests (d) and two-tailed Student’s t-tests (h). Critical significance thresholds were post hoc Šidák corrected.
a) Superimposed recordings of EPSCs evoked by pairs of stimuli at the calyx of Held at varying intervals (Δt) in 0.6 mM [Ca2+]e. b) Average paired-pulse ratios at the calyx of Held in 0.6 mM [Ca2+]e fit exponentially. c) Average paired-pulse ratios at Δt = 3 ms and time constant of exponential fits to data in b (P = 7.5 * 10−5). d) Representative EPSCs at the calyx of Held evoked by stimulation at 200 Hz. e) Average normalized EPSCs during 200 Hz stimulation. f) Normalized amplitude of the last 3 EPSCs (EPSC8-10) during 200 Hz stimulation (P = 3.3 * 10−5). g) Superimposed recordings of EPSCs evoked by pairs of stimuli at the calyx of Held at varying Δt in 0.3 mM [Ca2+]e. h) Paired-pulse ratios in climbing fibres in 0.3 mM [Ca2+]e fit exponentially. i) Average paired-pulse ratios at Δt = 10 ms and time constant of exponential fits to data in h (P = 5.5 * 10−7). j) Representative EPSCs evoked by climbing fibre stimulation at 50 Hz. k) Average normalized amplitudes of climbing fibre EPSCs during 50 Hz stimulation. l) Normalized amplitude of the last 3 EPSCs (EPSC8-10) during 50 Hz stimulation (P = 0.02). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as *: P < 0.05, **: P < 0.01, ***: P < 0.001. Significances were evaluated using Kruskal-Wallis tests (c) and two-tailed Student’s t-tests (b,e,f,h,i,l).
a) Superimposed recordings of EPSCs evoked by pairs of stimuli at the calyx of Held at varying intervals (Δt) in 0.6 mM [Ca2+]e. b) Average paired-pulse ratios at the calyx of Held in 0.6 mM [Ca2+]e fit exponentially. c) Average paired-pulse ratios at Δt = 2 ms and time constant of exponential fits to data in b (P = 0.001). d) Representative EPSCs at the calyx of Held evoked by stimulation at 200 Hz. e) Average normalized EPSCs during 200 Hz stimulation. f) Normalized amplitude of the last 3 EPSCs (EPSC8-10) during 200 Hz stimulation (P = 0.004). g) Best fit to WT and KO data of the biophysical model of SYT3-dependent vesicle docking (Extended Data Fig. 7d). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as **: P < 0.01. Significances were evaluated using two-tailed Student’s t-tests (b, e) and Kruskal-Wallis tests (c, f).
a) Schematic of cerebellar parallel fibre recordings. EPSCs were recorded from voltage-clamped Purkinje cells (PC) while parallel fibres were activated by electrical stimulation in the molecular layer. b) Superimposed recordings of EPSCs evoked by pairs of stimuli at the at varying intervals (Δt) in WT (black), Syt3 KO (magenta) and Syt7 KO (red) synapses. c) Average paired-pulse ratios fit exponentially. d) Average paired-pulse ratios at Δt = 5 ms and time constant of exponential fits to data in b. e) Representative EPSCs evoked by 20 stimuli at 50 Hz. f) Average normalized EPSCs during 50 Hz stimulation. Magenta and red bars indicate significant differences between WT and Syt3 KO, or WT and Syt7 KO, respectively. Gray bar indicates significant differences between Syt3 KO and Syt7 KO. g) Normalized amplitude of the last 5 EPSCs (EPSC16-20) during 50 Hz stimulation (WT vs. Syt3 KO: P = 0.0002; WT vs. Syt7 KO: P = 0.009; Syt3 KO vs. Syt7 KO: P = 0.007). Data are mean ± s.e.m. Number of experiments are shown in Extended Data Table 1. Statistical significances are shown as *: P < 0.05, **: P < 0.01, ***: P < 0.001. Significances were evaluated using one way ANOVA followed by two-tailed Student’s t-tests. Critical significance thresholds were post hoc Šidák corrected.
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Weingarten, D.J., Shrestha, A., Juda-Nelson, K. et al. Fast resupply of synaptic vesicles requires synaptotagmin-3. Nature 611, 320–325 (2022). https://doi.org/10.1038/s41586-022-05337-1